Emerging new evidence from both human studies and animal models implicate endothelin (ET) participation in variety of pathogenesis, including myocardial fibrosis. Cardiac fibrosis is characterized by an increased accumulation of type I collagen. It has been demonstrated that the activated (phenotypically transformed) fibroblast-like cells termed "myofibroblasts" (myoFb), which appear at the site of injury are responsible for expressing contractile protein (alpha smooth muscle actin) and type I collagen during tissue repair and remodeling. Recently, we have identified that myoFb isolated from the site of myocardial infarction, express high levels of TGFbeta/1 and also serve as a non-endothelial source of ET. The mechanisms involved in regulating ET production and its induction of extracellular matrix collagen gene expression by these cells is unclear. Proposed investigations will provide important insights into the new emerging pathophysiological roles of this vasoactive peptide. We will use myoFb to study the specific cellular signaling and molecular mechanisms of endothelin expression and its mechanism of action, including its possible cross-regulation with TGFbeta/1 in modulating collagen gene expression. An understanding of the complex cascade of cellular and molecular mechanisms of ET peptide may have important therapeutic implications. The specific aims are as follows: 1. to confirm de novo ET generation in myoFb, including its regulation of expression by ET itself or by TGF-beta/1; 2. to identify cis-acting elements and their trans- acting factors in the promotion of type I collagen [alpha1(I)] gene that respond to ET and TGFbeta/1. In these studies, the specific sequences involved in the alpha1(I) promoter will be identified by reporter gene assays; 3. to examine ET-receptor signaling pathway(s), particularly protein kinase C (PKC) and protein tyrosine kinase (PTK) involved in the induction of collagen gene expression; and 4. to examine the relationship between ET and TGF-beta/1 in the type I collagen expression induced by ET. To achieve this, we will investigate the role of trans-acting factors for ET and TGF-beta/1 in the alpha1(I) expression. The long term goals of this research is to understand the emerging new effects of ET and its interplay with growth factors such as TGF-beta/1 in cardiac tissue repair and pathologic remodeling that accompanies various forms of heart disease.